We have cloned and characterized gene fragments encoding epitopes to which sporozoite-neutralizing antibodies are directed. Recombinant proteins and synthetic peptides containing these epitopes are useful for the ability to induce an antigenic response or protection following immunization.
Cryptosporidiosis, caused by the protozoal agent Cryptosporidium parvum, has emerged as an important enteric disease of humans and animals. C. parvum was originally described in 1912, but not recognized as a human pathogen until 1976 [Tyzzer, E. E., Arch. Protistenkd., 26, 394-412 (1912); Meisel, J. L. et al., Gastroenterol., 70, 1156-1160 (1976); Nime, F. A. et al., Gastroenterol., 70, 592-598 (1976)]. The subsequent emergence of AIDS revealed the devastating impact of opportunistic infectious agents, including C. parvum, in immunocompromised hosts.
C. parvum infects intestinal epithelial cells and induces diarrhea, the duration and severity of which are determined by the immunological competency of the host [Current et al., N Engl. J. Med. 308, 1252-1257 (1983); Fayer and Ungar, Microbiol. Rev. 50, 458-483 (1986); O""Donoghue, Int. J. Parasit. 25, 139-195 (1995)]. Immunocompetent persons become infected following ingestion of a few hundred C. parvum oocysts [DuPont, H. L. et al., N. Engl. J. Med., 332, 855-859 (1995)]. Diarrhea lasts 1 to 14 days and ceases with the clearance of infection, a process dependent on CD4+ T lymphocytes [Ungar, B. L. P. et al., J. Immunol., 147, 1014-1022 (1991); Aguirre, S. A. et al., Infect. Immun., 62, 697-699 (1994); Perryman, L. E. et al., Infect. Immun., 62, 1474-1477 (1994)]. Immunocompetent hosts that recover from infection are generally resistant to reinfection. In contrast, immunodeficient hosts lacking sufficient CD4+ T lymphocytes develop persistent infections accompanied by severe, life-threatening diarrhea [See, e.g., Petersen, C., Clin., Infect. Dis., 15, 903-909 (1992); Flanigan, T. et al., Ann. Int. Med., 116, 840-842 (1992)]. The management of these patients is complicated by absence of efficacious drugs to control parasite replication [O""Donoghue, supra]. Consequently, investigators have evaluated oral administration of sporozoite- and merozoite-neutralizing antibodies to resolve C. parvum infection in hosts lacking sufficient CD4+ T lymphocytes [Watzl, B. et al., Am. J. Trop. Med. Hyg., 48, 519-523 (1993); Fayer, R. et al., J. Parasitol., 75, 151-153 (1989); Fayer, R. et al., J. Parasitol., 75, 393-397 (1989); Fayer, R. et al., Infect. Immun., 58, 2962-2965 (1990); Doyle, P. S. et al., Infect. Immun., 61, 4079-4084 (1993); Riggs, M. W. et al., Infect. Immun., 62, 1927-1939 (1994); Cama, V. A. and Sterling, C. R., J. Protozool., 38, 42S-43S (1991); Arrowood, M. J. et al., Infect. Immun., 57, 2283-2288 (1989); Bjorneby, J. M. et al., Infect. Immun., 59, 1172-1176 (1991); Perryman, L. E. et al., Infect. Immun., 58, 257-259 (1990); Perryman, L. E., et al., Infect. Immun., 61, 4906-4908 (1993); Tilley, M. et al., Infect. Immun., 59, 1002-1007 (1991).
The demonstrated importance of the immune response in prevention and recovery from C. parvum infection has stimulated investigators to define protective immune mechanisms and the characteristics of antigens which induce protective responses. Monoclonal antibodies C6B6, 17.41, 18.44, and 5C3 have been shown to diminish infection by C. parvum in neonatal mice. C6B6 reacts with p23; mAb 17.41 reacts with carbohydrate moieties on glycoproteins of 28 to 98 kDa; mAb 18.44 binds to a glycolipid conjugate termed CPS 500; and mAb 5C3 defines glycoproteins of 15 and 60 kDa [Arrowood, M. J. et al., Infect. Immun., 57, 2283-2288 (1989); Tilley, M. et al., Infect. Immun., 59, 1002-1007 (1991); Arrowood, M. J. et al., J. Parasitol., 77, 315-317 (1991); Riggs, M. W. et al., J. Immunology, 143, 1340-1345 (1989)]. Other partially protective mAb react with sporozoite proteins and glycoproteins ranging in molecular mass from 25 to  greater than 900 kDa (unpublished observations).
A first aspect of the present invention is an isolated DNA molecule comprising a nucleotide sequence encoding Cryptosporidium parvum p23 protein or an antigenic fragment thereof.
A further aspect of the present invention is an isolated DNA molecule encoding a peptide comprising amino acid sequence Gln Asp Lys Pro Ala Asp Ala Pro Ala Ala Glu Ala Pro Ala Ala Glu Pro Ala Ala Gln Gln Asp Lys Pro Ala Asp Ala) (SEQ ID NO:4).
A further aspect of the present invention is a method of inducing an antigenic response to Cryptosporidium parvum, by administering an antigenic protein or peptide according to the present invention, in an amount sufficient to induce an antigenic response.
A further aspect of the present invention is an antigenic peptide comprising the amino acid sequence: Gln-Asp-Lys-Pro-Ala-Asp (SEQ ID NO:9).
A further aspect of the present invention is a method of inducing an antigenic response to Cryptosporidium parvum, by administering to a subject an antigenic peptide comprising the amino acid sequence: Gln-Asp-Lys-Pro-Ala-Asp (SEQ ID NO:9), in an amount sufficient to induce an antigenic response in said subject.
A further aspect of the present invention is a monoclonal antibody that binds to the synthetic peptide Gln-Asp-Lys-Pro-Ala-Asp (SEQ ID NO:9).
A further aspect of the present invention is a method of providing passive immunity to Cryptosporidium parvum to a subject, by administration of a monoclonal antibody that binds to the synthetic peptide Gln-Asp-Lys-Pro-Ala-Asp (SEQ ID NO:9).
A further aspect of the present invention is a method of providing passive immunity to Cryptosporidium parvum to a subject, by administration of polyclonal antibodies that specifically bind to a peptide comprising the amino acid sequence: Gln-Asp-Lys-Pro-Ala-Asp (SEQ ID NO:9), in an amount sufficient to produce passive immunity in the subject.
Nucleotide sequence data reported in this work are available from the GenBank(trademark) data base with the accession number U34390.